Objective: To investigate the IL-10+CD19+ regulatory B cells (Breg) in peripheral blood of patients with rheumatoid arthritis (RA), and analyze the correlation between the expression of IL-10+CD19+ Breg cells and disease severity. Methods: A total of 40 patients with active RA and 30 healthy individuals (healthy controls, HC) were involved in this study. The peripheral blood mononuclear cells (PBMC) were isolated. The isolated PBMC were co-cultured with CpG oligodeoxynucleotide 2006 (CpG ODN 2006) and phorbol myristate acetate (PMA) in vitro. Flow cytometry was employed to analyze the expression of Breg before and after stimulation. The correlations of Breg expression with ESR, CRP and DAS28 were analyzed. Results: Before stimulation in vitro, the expression of Breg in PBMC of RA group and HC group were 1.92%(1.58%, 2.56%) and 2.04%(1.73%, 2.93%) respectively. There was no significant difference between the two groups (P=0.258). After induction in vitro, the expression of Breg of RA group [13.00%(9.75%, 14.85%)] was significantly higher than that of HC group [9.12%(6.83%, 10.22%)], P＜0.001. Spearman correlation analysis showed that Breg expression was negatively correlated with DAS28 (P=0.002), but not with ESR and CRP (P＞0.05). Conclusion The expression of Breg was significantly increased after stimulation in vitro and negatively correlated with DAS28, which indicated Breg may play important regulatory roles in pathogenesis and development of RA.

Objective: To quantify the serum levels of apoptosis-related molecules M30 and M65 in the patients with primary biliary cholangitis (PBC) and analyze their clinical significance for PBC. Methods: A total of 79 patients with PBC and 40 healthy individuals were involved in this study from January 2017 to April 2019. The levels of serum M30 and M65 were measured by enzyme-linked immunosorbent assay (ELISA) and the ratio of M30/M65 was calculated. The parameters of liver function were tested by automatic biochemical analyzer. ALT, AST and GGT were determined by rate method, ALP was assayed by the method of NPP substrate-AMP buffer and T-Bil was determined by oxidation method. Autoantibodies including AMA-M2, anti-GP210 and anti-SP100 were detected by automated multiplexed bead-based and immunoblotting assays. The correlation of M30, M65 and M30/M65 ratio with liver function items were analyzed by spearman assay. The levels of M30, M65 and M30/M65 ratio were analyzed by ROC curve to evaluate their values in PBC screening. Results: M65 level in PBC group was significantly higher than that in control group (P＜0.001) with statistically significant difference and M30/M65 ratio was significantly lower than that in control group with statistically significant difference (P＜0.001). There was no significant difference for the level of M30 between the two groups (P=0.641). Among the PBC patients, 18 were anti-GP210-positive and 17 were anti-SP100-positive. In anti-GP210 positive group the levels of M30 and M65 were significantly higher than those in the negative group (P values were 0.002 and 0.001 respectively). In anti-SP100 positive group the level of M65 was significantly higher than that in the negative group (P=0.027) and M30/M65 ratio was significantly lower than that in negative group with statistically significant difference (P=0.005). Spearman correlation analysis showed that the levels of M30 and M65 were positively correlated with ALT, AST and T-Bil (P＜0.05). The cut off values of M30 and M65 were 63.62 U/L and 155.37 U/L and M30/M65 was 0.32, respectively. The screening sensitivities were 63.30%, 89.10% and 93.30%, and the specificities were 51.60%, 90.00% and 75.00%, respectively. Conclusion The level of M65 in the serum of PBC patients significantly increased and M30/M65 ratio significantly decreased, which could be used as a promising serum marker of laboratory for PBC screening.

Objective To evaluate the changes of adenosine metabolism pathway related molecules and their contribution to inflammatory injury in primary biliary cholangitis (PBC).Methods The consecutive samples of 49 subjects with PBC from The First People's Hospital of Taicang and The Second People's Hospital of Changshu were recruited from October 2016 to October 2017,and 36 healthy controls were involved in this study.The expression of CD39 and CD73 on CD4+T cells and Foxp3 + regulatory T cells were assayed by flow cytometry and the concentration of adenosine triphosphate (ATP) in serum was analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS).The correlations between Tregs,ATP and liver function were analyzed,i.e.,alanine aminotransferase (ALT),aspartate aminotransferase (AST),gamma-glutamyltransferase (GGT),alkaline phosphatase (ALP) and Mayo scores.Results In the patients with PBC,low proportions of CD4+CD39+T cells were noted compared with healthy controls [(5.28 ± 1.92) ％ vs (11.0l ± 3.19) ％,t =10.25,P ＜ 0.01].The patients with PBC also had significantly low proportion of CD4+CD25 + Foxp3+ CD39+ T cells compared with healthy controls [(23.75 ± 9.48) ％ vs (54.68 ± 5.18) ％,t =13.79,P ＜0.01].No significant difference of the proportion of CD4+CD73+T or CD4+CD25+ Foxp3+CD39+T cells was found between PBC and control groups (t values were 2.235 and 1.083,P ＞ 0.05).The level of serum ATP was higher in the patients with PBC than that of healthy controls [(200.28 ± 79.41) μg/L vs (89.20 ± 33.76) μg/L,t =8.367,P ＜ 0.01].A significant correlation was demonstrated between the proportion of CD39 + Treg in total Treg cells and the levels of ATP (r =-0.413,P =0.003),GGT (r=-0.378,P=0.007) and Mayo score (r=-0.382,P=0.007).Conclusion The low proportion of CD39+ Treg cells may contribute to the down-regulation of ATP hydrolysis in the patients with PBC.No significant change of CD73 + Treg cells was found in PBC patients.

Objective To investigate clinical significance of serum retinol -binding protein(RBP)in chron-ic hepatitis B(CHB)and liver cirrhosis after hepatitis B infection(LC).Methods 109 hospitalized patients were involved in this study.They were 51 patients with CHB,58 patients with LC.56 healthy individuals were selected as the the health controls(HC).RBP,ALT,AST,GGT,LDH,TBIL,PA and ALB were analyzed by automatic biochemical analyzer.Results Decreased serum RBP was observed in patients with CHB and LC(CHB vs.HC:t =8.06,P <0.01;LC vs.HC:t =10.26,P <0.01).In addition,the concentration of serum RBP in group of LC was lower than that in CHB(t =3.41,P <0.01).RBP was positively correlated with PA in patients with CHB and LC(CHB:r =0.856,P <0.01;LC:r =0.737,P <0.01),and RBP was positively correlated with ALB in patients with CHB and LC (CHB:r =0.571,P <0.01;LC:r =0.328,P <0.05).ROC analysis showed that area under the receiver operator characteristic curve(AUC)in CHB and LC were 0.874 and 0.942,respectively.Conclusion Serum RBP decreases significantly in patients with CHB and LC.Serum level of RBP is associated with severity of liver diseases and maybe a potential prognostic index for liver disease.

Objective To investigate the expression of chemokine ligand 2 (CCL2)in chronic uric acid nephropathy(CUAN)and its diagnostic values in kidney damage.Methods 29 patients with CUAN[male 23,female 6,age (44.4 ±8.8)years old ]and 35 health individuals[male 27,female 8,age (40.6 ±7.8 )years old ]were involved in this study.Serum and peripheral blood mononuclear cells were isolated from peripheral blood.CCL2 was assayed by ELISA,and CD +45 /CD +14 monocytes were analyzed by flow cytometry.Liver &kidney functions,lipids and glucose were detected by automatic biochemistry analyzer.Results Serum CCL2 in group of CUAN and health con-trols were 456.2(202.6 -594.9)pg/mL and 245.0(132.2 -544.5)pg/mL,respectively(F =4.915,P =0.030). Percentages of monocytes in each group were 7.4%(5.6% -8.7%)and 6.1%(4.7% -7.9%),(F =8.891,P =0.004).Pearson analysis found that levels of CCL2 positively correlated with percentages of monocytes,serum uric acid and creatinine in CUAN group(r values were 0.535,0.584 and 0.012;P values were 0.003,0.001 and 0.012, respectively),but there was no correlation with urea and retinol binding protein(r value were 0.145 and 0.746,P val-ues were 0.453 and 0.453).Conclusion Hyperuricaemia may directly contribute to elevate levels of CCL2 and facilitate monocytes release into inflammation part to induce kidney damage.

Objective To investigate the population and role of IL-10+ CD19+ regulatory B cell (Breg) in patients with chronic hepatitis B.Methods Patients with acute hepatitis B (AHB) (n =28),chronic hepatitis B (CHB) (n =31) and normal subjects (n =25) were collected from Changshu No.2 People's Hospital between 2011 June and 2012 October.Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with CpG ODN 2006 and PMA.Flow cytometry was used to analyze the population of IL-10-CD19 + Breg,CD4 + CD25high Treg,and ELISA was used to analyze the concentration of IL-10 in culture supernatant.Results The population of Breg in Peripheral blood of the CHB group [1.28％ (1.05％-2.20％)] was higher than that in the AHB group [0.87％(0.55％-1.22％)] and the HCs group [0.89％ (0.51％-1.37％)] (P =0.001,0.006),and the difference between the AHB group and the HCs group was not statistically significant (P=0.669).Breg in the CHB group [14.30％ (10.70％-16.70％)] was higher than that in the AHB group [10.30％ (7.05％-13.30％)] and the HCs group [10.40％(6.85％-12.60％)] (P =0.003,0.001),treg in the CHB group [5.80％ (4.20％-9.10％)] was also higher than that in the AHB group [4.05％ (2.53％-5.40％)] and the HCs group [4.50％ (2.55％-5.50％)] (P ＜0.001,P =0.005),and there was no significantly difference between the AHB group and the HCs group (Breg:P =0.796 ; Treg:P =0.227).Spearman correlation analysis showed that Breg was positively correlated with Treg in the CHB group (r =0.50,P =0.004),however there was no significantly correlation in the AHB group and the HCs group (r =-0.15,P =0.462; r =0.09,P =0.669).The concentration of IL-10 in the CHB group was higher than that in the AHB group and the HCs group (P ＜ 0.001),and the difference between the AHB group and the HCs group was not statistically significant (P=0.341).Spearman correlation analysis showed that IL-10 were positively correlated with the population of Breg in the CHB group (r =0.409,P =0.022).Conclusion The poluations of regulatory B cell and regulatory T cell increased in patients with chronic hepatitis B,and Breg cell might play the immune regulation role through secreting IL-10 in chronic HBV infection.

Objective To investigate the role of interleukin-27 (IL-27) in proliferation and differentiation of CD4+ T cells in ankylosing spondylitis(AS).Methods CD4+ T cells were separated from peripheral blood which collected from AS patients and health controls(HCs).Cells proliferation was detected by CCK-8 kit,and cytokines were analyzed by ELISA.Real-time PCR was used to determine the mRNA expression of T-bet and GATA3.Results Serum IL-27 level in patients with AS was obviously higher than that in HCs(P ＜0.01).The proliferation rate of CD4+ T cells and the level of IFN-γin cultured medium in AS were higher than those in HCs group after IL-27 stimulation (P ＜ 0.01).IL-27 could induce T-bet mRNA expression in CD4+ T cells in AS (t =14.3,P ＜ 0.01),but no change was found in GATA3 mRNA expression.Conclusions IL-27 could induce the proliferation of CD4+ T cells and the activation of T-bet pathway in AS.Furthermore,Th1 immune response and related cytokines could be induced by IL-27 in AS.These implicate that IL-27 may play an important role in AS related inflammation.

Objective To investigate the correlation between immune inflammation and overactivity of T helper cells in childhood asthma by cell proliferation assay and activation induced cell death in vitro.Methods Th1/Th2/Th17 cytokines were determined by cytometric bead array.Cell proliferation and activation induced cell death were detected when CD4+ T cells were purified by magnetic beads and stimulated by PHA and antiCD3.At last,mRNA of Fas,FasL and Bcl-2 were mesured by real-time PCR.Results Cytokines of IL-4(2.451± 1.052ng/L vs 1.796 ±0.615 ng/L,P =0.018),IL-10( 1.920 ±0.813ng/L vs 1.390 ±0.162ng/L,P =0.006)and TNF(5.112 ±5.842 ng/L vs 1.506 ±0.551 ng/L,P =0.009) in sera of asthma group were higher than those in control group.Compared to control group,proliferation ability of CD4 + T cells in asthma group was greater ( OD450:0.498 ± 0.052 vs 0.274 ± 0.032,P ＜ 0.001 ) and apoptosis rate was lower( 35.62 ± 0.05 ％ vs 65.28±3.85％,P ＜0.001 ).mRNA expression of Fas in asthma group was lower but Bcl-2 was higher than those in control group.Conclusion It is implicated that defective expression of Fas and over expression of Bcl-2 in CD4+ T cells may contribute to apoptosis inhibition and cell proliferation,which could explain overeactivity of CD4 + T cells and lvmphocvte infiltration in childhood asthma.

Objective To investigate a new therapeutic pathway for primary biliary cirrhosis (PBC) by immune tolerance reestablishment in a PBC mouse model. Methods Spleenic cells from naive mice were incubated with M2 in the presence of ECDI and the ceils were injected into caudal vein of the mice which would be used for development of PBC model. Spleenic cells incubated with bovine serum albumin (BSA) were injected as controls. 16 weeks later, anti-mitoehondrial antibody (AMA) , alkaline phosphatase(AKP) and portal inflammation were assayed for evaluating the prevention effect. Results AMA positive rate in tolerance group was lower than that in BSA and PBC groups ( P = 0. 007, P = 0. 003 ). The difference between BSA and PBC was not significantly. Serum AKP levels in tolerance, BSA and PBC group were (80.5 ±9.8) U/L, (93.8 ±15.7) U/L and (92.5 ±17.7) U/L, separately. The level in tolerance group was lower than that in BSA and PBC groups (P =0.0095, P =0.029). The rates of portal areas with cell infiltration were 42. 67% ± 12. 30% , 57. 07% ± 11. 35% and 51. 53% ± 9. 96% , separately. The number of infiltrated portal tracts in tolerance group was less than that in PBC group (P = 0.039) and BSA group (P = 0. 0024). Conclusion PBC was prevented to some extent by reestablishing immune tolerance to M2 autoantigen. This provides clues for finding a better treatment proposal.

Objective To investigate the roles of TLR3 pathway activiated by polyI:C in proliferation and apoptosis of multiple myeloma (MM) RPMI8226 cell line.Methods RPMI8226 cells were cultured in RPMI 1640 with different dose of polyl:C.Cells were collected in different time.Proliferation and apoptosis were detected by CCK-8 kit and flow cytometry,separately.Results The proliferation of RPM18226 was inhibited by polyI:C,and it was dose and time dependent,24 h:12.30％ ±2.04％,22.50％±2.20％,37.90％ ±1.30％ ; 48 h:17.80％ ±1.52％,29.60％ ±0.85％,45.80％ ±1.68％ ;72 h:25.10％±1.01％,34.60％±1.27％,60.50％±2.08％,P＜0.05.RPMI8226 cells were incubated with 50 μg/ml,100 μg/ml and 200 μg/ml polyI:C for 48 h.Apoptotic rate were 5.60％ ±1.06％,8.71％ ±1.06％ and 13.93％ ±1.17％,P＜0.05.TLR3 and TRIF mRNA expression increased obviously and dose dependent,TLR3:1.41±0.10,2.24±0.16,4.08±0.13; TRIF:1.07±0.16,1.97±0.13,3.56±0.19,P＜0.05.Conclusion The proliferation of MM cells were inhibited by TLR3 pathway obviously,and apoptosis was induced by polyI:C.